KIR3DS1 directs NK cell-mediated protection against human adenovirus infections.

Human adenoviruses (HAdVs) are a major cause for disease in children, in particular after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, effective therapies for HAdV infections in immunocompromised hosts are lacking. To decipher immune recognition of HAdV infection and determine new targets for immune-mediated control, we used an HAdV infection 3D organoid system, based on primary human intestinal epithelial cells. HLA-F, the functional ligand for the activating NK cell receptor KIR3DS1, was strongly up-regulated and enabled enhanced killing of HAdV5-infected cells in organoids by KIR3DS1+ NK cells. In contrast, HLA-A and HLA-B were significantly down-regulated in HAdV5-infected organoids in response to adenoviral E3/glycoprotein19K, consistent with evasion from CD8+ T cells. Immunogenetic analyses in a pediatric allo-HSCT cohort showed a reduced risk to develop severe HAdV disease and faster clearance of HAdV viremia in children receiving KIR3DS1+/HLA-Bw4+ donor cells compared with children receiving non­KIR3DS1+/HLA-Bw4+ cells. These findings identify the KIR3DS1/HLA-F axis as a new target for immunotherapeutic strategies against severe HAdV disease.

The Loss of HLA-F/KIR3DS1 Ligation Is Mediated by Hemoglobin Peptides.

The human leukocyte antigen (HLA)-Ib molecule, HLA-F, is known as a CD4+ T-cell protein and mediator of HIV progression. While HLA-Ia molecules do not have the chance to select and present viral peptides for immune recognition due to protein downregulation, HLA-F is upregulated. Post HIV infection, HLA-F loses the affinity to its activating receptor KIR3DS1 on NK cells leading to progression of the HIV infection. Several studies aimed to solve the question of the biophysical interface between HLA ligands and their cognate receptors. It became clear that even an invariant HLA molecule can be structurally modified by the variability of the bound peptide. We recently discovered the ability of HLA-F to select and present peptides and the HLA-F allele-specific peptide selection from the proteomic content using soluble HLA (sHLA) technology and a sophisticated MS method. We established recombinant K562 cells that express membrane-bound HLA-F*01:01, 01:03 or 01:04 complexes. While a recombinant soluble form of KIR3DS1 did not bind to the peptide-HLA-F complexes, acid elution of the peptides resulted in the presentation of HLA-F open conformers, and the binding of the soluble KIR3DS1 receptor increased. We used CD4+/HIV- and CD4+/HIV+ cells and performed an MS proteome analysis. We could detect hemoglobin as significantly upregulated in CD4+ T-cells post HIV infection. The expression of cellular hemoglobin in nonerythroid cells has been described, yet HLA-Ib presentation of hemoglobin-derived peptides is novel. Peptide sequence analysis from HLA-F allelic variants featured hemoglobin peptides as dominant and shared. The reciprocal experiment of binding hemoglobin peptide fractions to the HLA-F open conformers resulted in significantly diminished receptor recognition. These results underpin the molecular involvement of HLA-F and its designated peptide ligand in HIV immune escape.

HLA-F on Autologous HIV-Infected Cells Activates Primary NK Cells Expressing the Activating Killer Immunoglobulin-Like Receptor KIR3DS1.

HIV-exposed seronegative KIR3DS1 homozygotes have a reduced risk of HIV infection. HLA-F is the ligand for the activating NK cell receptor (NKR) KIR3DS1. HLA-F is expressed on HIV-infected CD4 T cells. Coculture of sorted, HIV-infected CD4- (siCD4-) T cells with NK cells activated a higher frequency of KIR3DS1+ than KIR3DS1- NK cells from KIR3DS1 homozygotes to elicit anti-HIV functions such as CCL4, gamma interferon (IFN-γ), and CD107a expression. This was the case whether KIR3DS1+/- NK cells were analyzed inclusively or exclusively by gating out NK cells coexpressing the NKRs, KIR2DL1/L2/L3, 3DL2, KIR2DS1/S2/S3/S5, NKG2A, and ILT2. Blocking the interaction of HLA-F on siCD4- cells with KIR3DS1 on exclusively gated KIR3DS1+ NK cells with KIR3DS1-Fc chimeric protein or an HLA-F-specific monoclonal antibody reduced the frequency of activated KIR3DS1+ cells compared to that under control conditions. KIR3DS1+ NK cell activation by HIV-infected CD4+ cells may underlie the reduced risk of KIR3DS1 homozygotes to HIV infection.IMPORTANCE This study investigated a mechanism that may underly epidemiological studies showing that carriage of the KIR3DS1 homozygous genotype is more frequent among HIV-exposed seronegative subjects than among HIV-susceptible individuals. Carriage of this genotype is associated with a reduced risk of HIV infection. The protective mechanism involves the interaction of HLA-F on CD4+ cells infected with replication-competent HIV with the activating NK receptor, KIR3DS1. This interaction leads to the activation of KIR3DS1+ NK cells for secretion of cytokines and chemokines with anti-HIV activity. Among these is CCL4, which binds and blocks CCR5, the coreceptor for HIV entry of HIV into new target cells. In the setting of an exposure to HIV, incoming HIV-infected cells expressing HLA-F rapidly activate KIR3DS1+ NK cells to elicit anti-HIV activity. Exclusive gating strategies and blocking experiments support the notion that the HLA-F/KIR3DS1 interaction is sufficient to activate NK cell functions.

The Emerging Roles of Human Leukocyte Antigen-F in Immune Modulation and Viral Infection.

Human leukocyte antigens (HLAs) play various critical roles in both innate and adaptive immunity through processes such as presenting antigens to T cells and serving as ligands for receptors expressed on natural killer (NK) cells. Among the HLA class I family, the clinical significance and biological function of HLA-F have been the least investigated and have remained elusive for a long period of time. Previous studies have revealed that HLA-F expression might be involved in various physiological and pathological processes, such as pregnancy, viral infection, cancer, transplantation, and autoimmune diseases. However, recent data have shown that, akin to other HLA family members, HLA-F molecules can interact with both activating and inhibitory receptors on immune cells, such as NK cells, and can present a diverse panel of peptides. These important findings pave new avenues for investigations regarding the functions of HLA-F as an important immune regulatory molecule. In the present review, we summarize the studies on the role of HLA-F in immune modulation, with a special emphasis placed on the roles of HLA-F and KIR3DS1 interactions in viral infection.

A Variant of a Killer Cell Immunoglobulin-like Receptor Is Associated with Resistance to PD-1 Blockade in Lung Cancer.

PURPOSE: PD-(L)1-blocking antibodies have clinical activity in metastatic non-small cell lung cancer (NSCLC) and mediate durable tumor remissions. However, the majority of patients are resistant to PD-(L)1 blockade. Understanding mechanisms of primary resistance may allow prediction of clinical response and identification of new targetable pathways. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells were collected from 35 patients with NSCLC receiving nivolumab monotherapy. Cellular changes, cytokine levels, gene expression, and polymorphisms were compared between responders and nonresponders to treatment. Findings were confirmed in additional cohorts of patients with NSCLC receiving immune checkpoint blockade. RESULTS: We identified a genetic variant of a killer cell immunoglobulin-like receptor (KIR) KIR3DS1 that is associated with primary resistance to PD-1 blockade in patients with NSCLC. This association could be confirmed in independent cohorts of patients with NSCLC. In a multivariate analysis of the pooled cohort of 135 patients, the progression-free survival was significantly associated with presence of the KIR3DS1 allele (HR, 1.72; 95% confidence interval, 1.10-2.68; P = 0.017). No relationship was seen in cohorts of patients with NSCLC who did not receive immunotherapy. Cellular assays from patients before and during PD-1 blockade showed that resistance may be due to NK-cell dysfunction. CONCLUSIONS: We identified an association of the KIR3DS1 allelic variant with response to PD-1-targeted immunotherapy in patients with NSCLC. This finding links NK cells with response to PD-1 therapy. Although the findings are interesting, a larger analysis in a randomized trial will be needed to confirm KIRs as predictive markers for response to PD-1-targeted immunotherapy.

Analysis of KIR3DP1 Polymorphism Provides Relevant Information on Centromeric KIR Gene Content.

Four killer cell Ig-like receptor (KIR) genes, collectively referred to as framework genes, characterize almost all KIR haplotypes. In particular, KIR3DL3 and KIR3DL2 mark the ends of the locus, whereas KIR3DP1 and KIR2DL4 are located in the central part. A recombination hot spot, mapped between KIR3DP1 and KIR2DL4, splits the haplotypes into two regions: a centromeric (Cen) region (spanning from KIR3DL3 to KIR3DP1) and a telomeric region (from KIR2DL4 to KIR3DL2), both varying in KIR gene content. In this study, we analyzed KIR3DP1 polymorphism in a cohort of 316 healthy, unrelated individuals. To this aim, we divided KIR3DP1 alleles into two groups by the use of a sequence-specific primer- PCR approach. Our data clearly indicated that KIR3DP1 alleles present on haplotypes carrying Cen-A or Cen-B1 regions differ from those having Cen-B2 motifs. Few donors (∼3%) made exceptions, and they were all, except one, characterized by uncommon haplotypes, including either KIR deletions or KIR duplications. Consequently, as KIR2DL1 is present in Cen-A and Cen-B1 regions but absent in Cen-B2 regions, we demonstrated that KIR3DP1 polymorphism might represent a suitable marker for KIR2DL1 gene copy number analysis. Moreover, because Cen-B1 and Cen-B2 regions are characterized by different KIR3DP1 alleles, we showed that KIR3DP1 polymorphism analysis also provides information to dissect between Cen-B1/Cen-B1 and Cen-B1/Cen-B2 donors. Taken together, our data suggest that the analysis of KIR3DP1 polymorphism should be included in KIR repertoire evaluation.

Killer-cell immunoglobulin-like receptors associated with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) affects the endocrine system and is associated with low-grade inflammation. Natural killer (NK) cells are involved in the defense of the female reproductive tract, folliculogenesis, ovulation and the menstrual cycle. The killer-cell immunoglobulin-like receptors (KIR) on the surface of NK cells modulate the activation and function of these cells after interacting with human leukocyte antigen (HLA) class I ligands. The objective of this study was to evaluate the possible association of the KIR and their HLA ligands with polycystic ovary syndrome. METHODS: Ninety-three patients with PCOS according to the Rotterdam criteria and 104 healthy controls were included in this study. The HLA class I and KIR genotypes were determined using a PCR-SSO technique, rSSO Luminex®. In order to assess whether the distribution of the HLA and KIR genotypes was in Hardy-Weinberg equilibrium, Arlequin 3.1 software was used. The frequency distributions in the two study groups were compared using the chi-squared statistic with Yates´s correction using Open Epi software. RESULTS: The higher frequencies of KIR3DS1-Bw4 (41% vs. 19%, Pc = 0.002; OR = 2.90) and homozygotic KIR2DS4-del (54% vs. 26%, Pc = 0.0002; OR = 3.316) in patients compared with controls suggest they confer susceptibility to PCOS. A lower frequency of KIR2DS4-full was observed in patients (43% vs. 70%, Pc = 0.0004, OR = 0.320). CONCLUSION: KIR and its HLA ligands were associated with the development of PCOS in the studied population.

KIR2DS1, 2DS5, 3DS1 and KIR2DL5 are associated with the risk of head and neck squamous cell carcinoma in Iranians.

BACKGROUND: Activating and inhibitory KIR receptors (aKIR, iKIR) control the development and function of NK cells whose function alterations adjust the tumor microenvironment immunity. This research was conducted to determine the KIRs gene impact on genetic predisposition to Head and Neck Squamous Cell Carcinoma (HNSCC) in Iranians. METHODS: KIR genotyping using sequence-specific primers-polymerase chain reaction (SSP-PCR) method was performed to identify the presence of all 16 KIR genes in 285 HNSCC patients, including laryngeal, oral cavity and pharyngeal SCC and 273 controls (CNs). RESULTS: Comparison of KIRs gene frequency between HNSCC and CNs revealed a highly significant increase in KIR2DL5, 2DS1, 2DS5, 3DS1 and CxT4 genotype and a decrease in KIR2DS4 deleted variant and AA genotype carriers. A significant increase was noted in individuals withhigher iKIRs than aKIRs in HNSCC compared with CNs. Individuals with ≥4 iKIR and those with ≥5 aKIRs were significantly more common in HNSCC than CNs. 68distinct KIR genotypes were identified in 558 individuals. CONCLUSION: Our findings determined the detrimental impact of KIR2DS1, 2DS5, 3DS1, 2DL5 and CxT4 genotype as well as the protective impact of KIR2DS4del and AA genotype on genetic predisposition to HNSCC in Iranians.

The novel KIR3DS1*082 allele identified in a southern Chinese Han individual.

KIR3DS1*082 allele differs from the closest allele KIR3DS1*01301 at nucleotide 1114T>C in exon 8.

The novel KIR3DS1*078 allele identified in a southern Chinese Han individual.

KIR3DS1*078 allele differs from the closest allele KIR3DS1*01301 at nucleotide 775 G>C in exon 5.

Killer immunoglobulin-like receptors can predict TKI treatment-free remission in chronic myeloid leukemia patients.

Several factors are predictive of treatment-free remission (TFR) in chronic myeloid leukemia (CML), but few data exist on the role of natural killer (NK) cells and their killer-cell immunoglobulin-like receptors (KIRs). KIR and human leukocyte antigen (HLA) genotypes were investigated in 36 CML patients who discontinued tyrosine kinase inhibitor (TKI) treatment after achieving deep molecular response (MR(4.5)). Cumulative TFR was significantly higher in patients homozygous for KIR A haplotype (85.7% vs. 45.5%; p = 0.029). Younger age, Bx haplotype, and the combination KIR3DS1/KIR3DL1 present/HLA-Bw4 present were significantly associated with relapse. KIR genotypes could prove useful in identifying patients that are likely to maintain MR(4.5) after discontinuing TKI treatment.

Peptide-Dependent Recognition of HLA-B*57:01 by KIR3DS1.

UNLABELLED: Killer cell immunoglobulin-like receptors (KIRs) play an important role in the activation of natural killer (NK) cells, which in turn contribute to the effective immune control of many viral infections. In the context of HIV infection, the closely related KIR3DL1 and KIR3DS1 molecules, in particular, have been associated with disease outcome. Inhibitory signals via KIR3DL1 are disrupted by downregulation of HLA class I ligands on the infected cell surface and can also be impacted by changes in the presented peptide repertoire. In contrast, the activatory ligands for KIR3DS1 remain obscure. We used a structure-driven approach to define the characteristics of HLA class I-restricted peptides that interact with KIR3DL1 and KIR3DS1. In the case of HLA-B*57:01, we used this knowledge to identify bona fide HIV-derived peptide epitopes with similar properties. Two such peptides facilitated productive interactions between HLA-B*57:01 and KIR3DS1. These data reveal the presence of KIR3DS1 ligands within the HIV-specific peptide repertoire presented by a protective HLA class I allotype, thereby enhancing our mechanistic understanding of the processes that enable NK cells to impact disease outcome. IMPORTANCE: Natural killer (NK) cells are implicated as determinants of immune control in many viral infections, but the precise molecular mechanisms that initiate and control these responses are unclear. The activating receptor KIR3DS1 in combination with HLA-Bw4 has been associated with better outcomes in HIV infection. However, evidence of a direct interaction between these molecules is lacking. In this study, we demonstrate that KIR3DS1 recognition of HLA-Bw4 is peptide dependent. We also identify HIV-derived peptide epitopes presented by the protective HLA-B*57:01 allotype that facilitate productive interactions with KIR3DS1. Collectively, these findings suggest a mechanism whereby changes in the peptide repertoire associated with viral infection provide a trigger for KIR3DS1 engagement and NK cell activation.

KIR3DS1*0130111: a novel KIR allele identified using molecular typing methods.

KIR3DS1*0130111 differs from KIR3DS1*0130101 with two previously undescribed single nucleotide polymorphisms.

Protective genotypes in HIV infection reflect superior function of KIR3DS1+ over KIR3DL1+ CD8+ T cells.

Certain human class I histocompatibility-linked leukocyte antigen (HLA)/killer cell immunoglobulin-like receptor (KIR) genotypic combinations confer more favourable prognoses upon exposure to human immunodeficiency virus (HIV). These combinations influence natural killer (NK) cell function, thereby implicating NK cells in protection from HIV infection or disease progression. Because CD8(+) T cells restrict HIV replication, depend upon HLA class I antigen presentation and can also express KIR molecules, we investigated how these HLA/KIR combinations relate to the phenotype and function of CD8(+) T cells from uninfected controls and individuals with chronic HIV infection. CD8(+) T cells from KIR3DL1 and KIR3DS1 homozygous individuals, and expressing the corresponding KIR, were enumerated and phenotyped for CD127, CD57 and CD45RA expression. Ex vivo and in vitro responsiveness to antigen-specific and polyclonal stimulation was compared between KIR-expressing and non-expressing CD8(+) T cells by interferon-γ production. There were higher numbers and fractions of KIR3DL1-expressing CD8(+) T cells in HIV-infected individuals independent of HLA-Bw4 co-expression, whereas expansion of KIR3DS1-expressing CD8(+) T cells reflected HLA-Bw4*80I co-expression. KIR3DL1(+) and S1(+) CD8(+) T cells were predominantly CD127(-)CD57(+)CD45RA(+). KIR3DL1-expressing CD8(+) T cells were insensitive to ex vivo stimulation with peptides from HIV or common viruses, but responded to anti-CD3 and recovered responsiveness to common viruses in vitro. Ex vivo non-responsiveness of KIR3DL1-expressing CD8(+) T cells was also independent of HLA-Bw4. KIR3DS1-expressing T cells responded normally to ex vivo antigenic stimulation, illustrating functional superiority over KIR3DL1(+) CD8(+) T cells.

Activating killer immunoglobulin-like receptors genes are associated with increased susceptibility to ankylosing spondylitis.

The aim of this study was to analyse the association of specific killer cell immunoglobulin-like receptors (KIR) genes and haplotypes with susceptibility to ankylosing spondylitis (AS) and its different clinical manifestations in a Spanish population. The presence or absence of all KIR genes was studied for their association with AS. A total of 176 patients with AS and 435 healthy control subjects were selected for this study based on clinical criteria. The commercial KIR-sequence-specific oligonucleotides (SSO) typing kit was used to investigate KIR typing. Frequencies of KIR2DS1 and KIR3DS1 genes were increased significantly in patients compared with healthy controls [52·8 versus 38·2%, PBonf < 0·01, odds ratio (OR) = 1·81 (1·28-2·59); 51·7 versus 37·5%, PBonf < 0·01, OR = 1·79 (1·25-2·54)]. Moreover, the frequency of activating genotypes in the AS patient group was significantly higher than in the healthy control group (P < 0·05). KIR2DS1 and KIR3DS1, in addition to human leucocyte antigen (HLA)-B27, may play an important role in the pathogenesis of AS. However, we show that the contribution of the KIR genes to AS susceptibility extends beyond the association with individual KIRs, with an imbalance between activating and inhibitory KIR genes seeming to influence the susceptibility to AS.

A novel KIR3DS1*0130107 allele isolated by sequencing from an Asian donor.

This novel KIR3DS1 allele officially named as KIR3DS1*0130107 was isolated from DNA samples from Asia using high-resolution sequenced-based techniques. KIR3DS1*0130107 differs from the first member of the KIR3DS1*013 subgroup (KIR3DS1*0130101) by a single mutation at position 8922A>G (intron 5), just nine nucleotides away from the start of exon 6.

Description of a novel KIR3DL1*0050104 allele identified using sequence-based techniques.

KIR3DL1*0050104 allele differs from KIR3DL1*0050101 at nucleotide positions 6709C>T (intron 5) and 11365A>G (intron 6).

Report of a novel KIR3DS1*0130104 allele discovered using advanced molecular techniques.

KIR3DS1*0130104 is identical to KIR3DS1*0130101 except for changes at positions 1768 (C>T) and 12617 (C>A).

A comprehensive analysis of the binding of anti-KIR antibodies to activating KIRs.

Analysis of killer cell immunoglobulin-like receptor (KIR) expression has been notoriously difficult because of the cross-reactivity of available antibodies, in particular between activating and inhibitory isoforms. We undertook a comprehensive study of available anti-KIR antibodies binding to activating KIRs (a-KIRs). Using cell lines stably transfected with a-KIRs (KIR2DS1-S5 and KIR3DS1), we confirmed documented binding specificities. In addition, we show that clones HPMA4 and 143211-previously assumed to be specific for KIR2DS1/L1 and KIR2DL1, respectively-bind KIR2DS5 and KIR2DS3 (HPMA4), and KIR2DS5 (143211). Other antibodies with previously undocumented binding were JJC11.6 (recognizing KIR2DS3) and 5.133 (recognizing all a-KIRs except KIR2DS1 and KIR2DS3). The novel KIR2DS5 reactivities were confirmed by blocking with soluble KIR-Fc fusion proteins, and by reverse transcriptase-PCR analysis of sorted primary natural killer cells. In conclusion, we show formerly undocumented binding properties of anti-KIR antibodies. These cross-reactivities should be taken into account when analyzing KIR expression.